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Odontogenic Keratocysts b, a.q-1 P53 AND K¡-67 (MlB-l) EXPRESSION IN ODONTOGENIC KERATOCYSTS Thesis submitted in partial fulfillment of the requirements for the degree of Master of Dental SurgerY Adi Rahmadsyah DDS (Medan) March 1998 Faculty of Dentistry The University of Adelaide I l This thesis is dedicated to Ayah & lbu Hanim, Sasha and Della TABLE OF CONTENTS Page Declaration Acknowledgements Chapter 1 lntroduction 1 Ghapter 2 Literature Review 4 2.1 The odontogenic keratocYst 5 2.1.1 Clinical features 6 2.1.2 Radiologicalfeatures 11 2.1.3 Histopathological features 12 2.1.4 Behaviour 16 2.1.5 Histogenesis & pathogenesis of odontogenic 18 keratocysts 2.2 P53 20 2.2.1 lnitial identification of P53 20 2.2.2 P53 structural characteristics 21 2.2.3 Molecular weight and location of p53 22 2.2.4 p53 protein level in cells 22 2.2.5 Types of p53 23 2.2.5.1 Wild-type p53 23 2.2.5.2 Mutant p53 24 2.2.6 Biological function of P53 27 2.2.6.1 Checkpoint of cell cYcle 28 2.2.6.2 Apoptosis 29 2.2.6.3 Cellular differentiation 29 2.2.7 Detection of p53 in Cells 30 2.2.8 Application of p53 in tumour diagnosis 30 2.2.9 P53 protein expression in oral lesion 33 2.3 Ki-67 and its functions 35 2.3.1 Ki-67 35 2.3.2 MIB-1 as an alternative to Ki-67 37 2.3.3 Ki-67lMlB-1 as indicators of cellular 39 proliferation 2.3.4 Expression of Ki-67 / MIB-1 in tumour cells 39 Chapter 3. Materials and Methods 43 3.1 Materials 44 3.1.1 Case selection 44 3.2 Methods 46 3.2.1 Preparation of histologic material 46 3.2.2. Assessment of clinical data and histological 47 material 3.2.2.1 Clinical data 47 3.2.2.1.1 Age 47 3.2.2.1.2 Sex 47 3.2.2.1.3 Site 47 3.2.2.2 Hlstological assessmerlI 48 3.2.2.2.1 Epithelial lining 48 3.2.2.2.2 Number of cysts and behaviour 48 3.2.2.2.3 lnflammation 48 3.2.4 lmmunohistochemical phase 49 Chapter 4 Results 50 4.1 Clinical data 51 4.1.1 Age distribution of total OK Pool 51 4.1.2 Age distribution of selected OK cases 51 4.1.3. Sex distribution of total OK pool subjects and 54 immunohistochemistry pool subjects 4.1.4 Site distribution of total OK Pool 54 4.2. Histologic obseruations 58 4.2.1 Epithelial lining 58 4.2.2 I mmunohistochemical staining 62 4.2.2.1 P53 staining 62 4.2.2.2 Relationship between p53 staining and 66 inflammation 4.2.2.3 MIB-1 staining 68 4.2.2.4 Relationship between p53 staining and MIB-1 70 positivity and inflammation 4.2.2.5 Relationship between p53 staining and MIB-1 72 staining 4.2.2.6 Ameloblastoma 74 Chapter 5 Discussion 77 5.1 Clinical and histological findings 78 5.2 Technical aspects of immunostaining 79 5.3 Pre-treatment effects on staining 81 5.4 p53 staining 82 5.5 MIB-1 staining 83 5.6 P53 staining and inflammation 83 5.7 MIB-1 staining and inflammation 84 5.8 Ameloblastoma 84 Conclusions 86 Bibliography 88 DECLARATION This work contains no material which has been accepted for the award of any other degree of diptoma in any university or tertiary institution and to the best of my knowledge and belief contains no material previously published or written by another person, except where due reference has been made in the text. I give consent to this copy of my thesis being made available for loan and photocopying Adi R h March 1998 ACKNOWLEDGEMENTS I am deeply grateful for the patience and support of Associate Professor David F. Wilson, the supervisor of this project. He has donated extraordinary amounts of time, and has provided much invaluable advice on aspects of scientific research and methodology. Many thanks also go to Dr Angela Pierce for her assistance and advice, and to Sandie Powell and Margaret Leppard for laboratory and photography assistance. Thanks especially to the Director and Staff of the lnstitute of Medical and Veterinary Science (IMVS), Adelaide for allowing access to laboratories as a r+ rrJ part of my training. Thanks also to Peter Gilan, IMVS for helping with the immunohistochemical staining of the specimens. A thankyou also to Professor Grant Townsend for his assistance in statistical analysis. Sincere thanks are also expressed to the Australian Agency for lnternational Development (AusAlD) for sponsoring me in this Master of Dental Surgery program. My thanks are extended to Ms Jo Seton, ACUE, University of Adelaide for invaluable assistance in editing draft materials. Special thanks also to Mrs. Juliefllugo for her help in the final preparation of this thesis. þ CHAPTER 1 INTRODUCTION 1 Odontogenic keratocysts (OKs) are developmental cysts of the jaws and are known to express growth and behavioural characteristics more analogous to benign neoplasms than to simple jaw cysts. A number of studies on odontogenic keratocysts have emphasized that the aggressive behavior of these cysts might be associated with epithelial cyst lining activities' :. i The unusual behavior and the characteristic histological features of OKs have been the subject of many studies. More recently the results of a number of immunohistochemical studies have been reported in the literature. For example a small number of studies have indicated that p53 (p53 protein is a product of the p53 gene and acts as a checkpoint which monitors the efficacy and completion of the obligatory events in the cell cycle and blocks uncontrolled cell division) expression in OKs is higher than in other jaw cysts and that the location of p53 positive cells was more likely in the suprabasal epithelial cells. However, in these studies it is apparent that there is considerable variability when the results of different investigations are compared. ln addition to studies concerning p53 distribution in OKs, there have been a very small number of studies investigating the presence and distribution of anothercell proliferation marker Ki-67 (MlB-1). To date the results of these few studies have indicated that OKs express Ki-67 (MlB-1) at a level greater than for p53. 2 Apart from recent studies by Slootweg (1995) who studied the distribution of p53 and Ki-67 in a sample of 13 OKs and Li et al (1996) who undertook a similar study on a sample of 22 OKs there are few data available describing the correlated distribution of p53 and Ki-67 (MlB-1) in OKs. The aims of the present study were to: . Analyse p53 and MIB-1 distribution in a large sample of oKs. Determine whether or not staining patterns correspond for each marker. A small sample (9) of ameloblastomas were also analysed' Fifty cases of OKs were examined for p53 and MIB-1 (Ki-67) expression using immunohistochemical techniques following microwave pretreatment of histological sections. The primary antibody used for immunohistochemical staining of OKs and ameloblastoma were monoclonal p53 (Dako-DO-7) and MIB-1 (Zymed). J CHAPTER 2 LITERATURE REVIEW 4 2.1. THE ODONTOGENIC KERATOCYST Odontogenic keratocysts (OKs) are developmental cysts of the jaws. The cysts are thought to originate from odontogenic epithelium, such as dental lamina or its remnants within bone or oral mucosa, and from the proliferation of basal cells of the oral epithelium (Wilson and Shear,1992i Browne, 1970b). Histologically, these cysts are characterised by keratin formation. The term odontogenic keratocyst was introduced by Philipsen (1956) and its essential clinical and radiological features were described by Pindborg and Hansen (1963). At first, the term was applied to any odontogenic cyst which had undergone keratinization. However, Hansen (1967) concluded that odontogenic keratocysts were not of inflammatory origin. Prior to classification of their terminology in the nineteen sixties OKs had long been known by different terms. ln his book Cysts of the Oral Region, Shear (1992) stated that this cyst was described as a dermoid cyst by Mikulicz (1876) and then as a cholesteatoma by Hauer (1926) and Kostecka (1929)' Although some authors had suggested the term "primordial cyst" as a synonymous term for odontogenic keratocyst, the former term is still not accepted worldwide because of the lack of evidence to support it' The term primordial cyst had been used to describe cysts developing in place of a tooth through degeneration of the enarnel organJhsterm was first used by Robinson (1945) and, subsequently, used by the World Health 5 Organization in 1971. ln 1985, Main and Shear suggested the term "odontogenic keratocyst" be used to designate this cyst of the jaws' Histologically, the OK has a characteristic appearance. lt can occur as a single cyst or multiple cysts. lt can also be associated with the Basal Cell Naevus Syndrome (BCNS). 2.1.1. Glinical features The clinical features of OKs, such aS age, incidence, Sex, the site of occurrence and the number and size of lesions are relatively variable. The incidence of OKs reported in studies conducted between the 1960s and 1990s also varies. Compared to other jaw cysts, the incidence of OKs is relatively low. A study by Toller (1967) reported that 33 (11.0%) of 300 jaw cysts of all types were OKs. ln contrast, a study by Hoffmeister and Harle (1985) showed that only 51 (1 .5 %) of 3353 cases of jaw cysts recorded were OKs. The incidence of OKs varies between white and black populations, with the white population more likely to suffer OKs than the black population, According to The World Standard Population, the incidence of OKs is higher in white males than in white females, black males and black females (Rachanis and Shear 1978).
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